Project Summary/Abstract: Cell-cell communication lies at the heart of evolution of multi-cellular life: cells send specific signals to instruct their neighbors to adopt fates distinct of their own. An important class of such developmental signals is encoded by the Wnt gene family. Wnt proteins interact with their cognate receptors of the Frizzled (Fzd) family to control countless developmental processes, from establishing the polarity of a single cell within a tissue to specifying the anterior-posterior body axis of an organism. Deregulation of Wnt signaling can have catastrophic consequences, including embryonic lethality, birth defects, and disease, most notably cancer. With their diverse and potent activities in development and disease, Wnts have captivated the attention of many researchers seeking to instruct and guide cellular fate choices. However, the basic mechanisms by which Wnt signaling achieves these highly varied developmental outcomes remain poorly understood and characterized. The long-term objective of the proposed research is to elevate the current understanding of the mechanisms by which Wnt proteins and their signaling pathways regulate stem cells and their ability to self-renew and differentiate. One of the main challenges in studying Wnt signaling and its roles in stem cell biology has been the scarcity of effective tools and assays to manipulate (activate and inhibit) and monitor Wnt signaling. Furthermore, the 19 Wnts and 10 Fzd proteins encoded in the mammalian genome interact with each other promiscuously, at least in ex vivo and in vitro cell-based systems, making it difficult to specifically affect individual signaling pathways. We have developed an innovative approach that utilizes engineered Wnt agonists, called Wnt mimetics, which exhibit superior biochemical properties compared to native Wnt proteins, to specifically target and activate individual Fzd receptors. Using human pluripotent stem (hPS) cells as a model system, the first Aim of this proposal will generate and characterize tools that will allow us to specifically activate individual FZD receptors, including multiple Wnt mimetics and gene edited hPS cell lines. In the second Aim, we will employ transcriptome- and proteome-wide approaches to examine to what extent pathway activation with FZD-specific Wnt mimetics differs from pathway activation with native Wnt proteins. This analysis will allow us to test our hypothesis that selective engagement of individual Wnt receptors triggers distinct signaling outputs and biological effects. Finally, in the third Aim, we will explore the role of a well-characterized human FZD7-specific Wnt mimetic in mice carrying a humanized Fzd7 gene. This approach will allow us to test Wnt mimetics in organoid cultures and whole animals. IMPACT: The proposed and innovative research will significantly advance the field of stem cell research by establishing new tools and methods to manipulate Wnt signaling in vitro and in vivo. With its abundant roles in human disorders and diseases, such as cancer, a better understanding of Wnt signaling is essential for the development of novel therapies for currently incurable diseases.